This creates an environment for increased rapid bioavail ability of E2 which can elicit nongenomic effects such as Ca2 mobilization, kinase activation, and alterations in dopamine subcellular location via membrane estrogen receptors. We have previously examined a well characterized this research non transfected neuronal cell culture model that expresses three known mERs mER, mER, and GPR30. in these cells physiological lev els of E2 and low levels of xenoestrogens can rapidly reverse actions of the DAT. Modifications Inhibitors,Modulators,Libraries in the phosphorylation state of the DAT by kinases causes alterations in the function and location of the DAT. Amphetamine, a psychostim ulant, also causes reversal and altered cellular location of the DAT which is known to be regulated by kinases, phos phatases, and Ca2 localization and association.

Therefore, we hypothesized Inhibitors,Modulators,Libraries that the estrogen mediated changes in dopamine efflux that we have observed may involve similar mechanisms. In this study we exam ined both indirect and direct mechanisms involved in physiological estrogen mediated dopamine efflux in Inhibitors,Modulators,Libraries con junction with the cellular location of the ERs and the DAT. We studied the involvement of protein kinases A and C, phospho inositol 3 kinase, extracellu lar regulated kinases , vesicular release of dopamine, and changes in intracellular Ca2 concentra tions in the actions of estrogens. Then we addressed the subcellular localization of ER, ER, the alternative mem brane ER, and DAT to see if estrogen induced trafficking of these proteins in and out of the plasma membrane could explain some of the regulatory effects on dopamine efflux.

In addition to E2, we also examined the effects of estrone and estriol to see if these estrogens may have some potent nongenomic sign aling effects Inhibitors,Modulators,Libraries of their own, as we have previously observed in pituitary cells, and if they can also affect DAT func tion. These differential regulatory effects on DAT by differ ent physiological estrogens may provide some insights into mechanisms controlling the incidence of neurologi cal diseases during life stages accompanied by fluctuations or change in the steady state levels of these hormones. Methods PC12 cell culture PC12 cells were grown in high glucose, phenol red free RPMI 1640 medium containing 5% fetal bovine serum and 5% equine serum. To promote PC12 dif ferentiation and minimize the effects of endogenous hor mones respectively, 20 ng ml NGF was added in medium supplemented with 0.

5% of 4 charcoal stripped FBS and HS for 48 hrs prior to experiments. Dopamine efflux assay We measured 3H dopamine Inhibitors,Modulators,Libraries efflux using selective catecho lamine transporter inhibitors to define specific dopamine transport via the DAT as previously described in. PC12 cells were plated on poly D lysine coated 48 well plates and uptake buffer containing neverless 0.